JP2006131595A - Carcinogenesis preventing agent and method for preparing cycloartane-type triterpene compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a safer and more effective carcinogenesis preventing agent particularly containing a cycloartane-type triterpene compound. <P>SOLUTION: The carcinogenesis preventing agent contains at least one cycloartane-type triterpene compound selected from the group consisting of 24-methylcycloartane-3β,24,24<SP>1</SP>-triol, 24<SP>1</SP>-methoxy-24-methylcycloartane-3β,24-diol, cycloartan-3,24-dione, 4,4,14-trimethyl-9,19-cyclopregnane-3,20-dione, 24,25-dihydroxycycloartan-3-one, 25-hydroxycycloart-23-en-3-one, and 25-hydroxy-24-methoxycycloartan-3-one. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a carcinogenesis-preventing agent and a method for producing a cycloartane-type triterpene compound, and more particularly to a carcinogenesis-preventing agent containing a cycloartane-type triterpene-type compound and a method for producing a cycloartane-type triterpene-type compound.

Conventionally, triterpene compounds have been known as carcinogenesis preventing agents (see Patent Document 1). This Patent Document 1 discloses a technique for obtaining several types of triterpene compounds effective for preventing carcinogenesis by chemically converting γ-oryzanol obtained by treating rice bran ingredients.
JP 2003-277269 A

However, in view of the great need for carcinogenesis prevention, there is a great expectation that a safer and more effective carcinogenic prevention agent cannot be obtained.
The present invention has been made in view of the above-described problems, and an object thereof is to provide a safe and effective carcinogenic preventive agent.

In order to solve the above-mentioned problems, the present inventors have focused on cycloartane-type triterpene compounds, which have been reported to have safety and excellent carcinogenicity prevention effects, and have further excellent carcinogenicity prevention effects. We conducted intensive research to search for compounds. As a result, it was found that by using filamentous fungi, a cycloartane-type triterpene compound having an excellent cancer prevention effect can be obtained. That carcinogenesis preventive agent according to claim 1 of the present invention, cyclo user Calais Nord, 24 methylcyclopentadienyl Altan-3.beta, 24, 24 ¹ - triol, 24 ¹ - methoxy-24-methyl-cyclopropyl Altan-3.beta, 24-diol, cyclo Artan-3,24-dione, 4,4,14-trimethyl-9,19-cyclopregnan-3,20-dione, 24,25-dihydroxycycloartan-3-one, 25-hydroxycycloalto-23 It contains at least one cycloartane type triterpene compound selected from the group consisting of ene-3-one and 25-hydroxy-24-methoxycycloartan-3-one.

If it is a cycloartane type triterpene mentioned above, it is excellent in the carcinogenesis prevention effect, and a draft tax is also high.
In addition, the compound of the present invention described above includes medically used and pharmacologically acceptable salts of each compound. Examples of such salts include alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, iron salt, zinc salt and copper salt. Metal salts such as nickel salts and cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine Salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, Tris (Hydroxyme And an amino amine salt such as glycine salt, lysine salt, arginine salt, ornithine salt and asparagine salt.

Moreover, when the compound of this invention of Claim 1 mentioned above forms a solvate (for example, hydrate), all these are also contained in this invention. For example, when the compound of the present invention is left in the air or recrystallized, it may absorb moisture, adsorbed water may adhere, or form a hydrate. Such solvates are also included in the present invention.
Furthermore, the compound of the present invention described in claim 1 has several asymmetric carbon atoms, and therefore various optical isomers exist. In the present invention, unless otherwise specified, all of these isomers including racemic compounds and mixtures of these isomers are included.

In addition, the anti-carcinogenic agent according to claim 2 of the present invention is that according to claim 1, wherein the cycloartane-type triterpene compound is obtained from a microbial conversion reaction by filamentous fungi using a compound obtained by treating rice bran ingredients as a substrate. It is a reaction product or a reaction product obtained by a chemical conversion reaction of a compound obtained by treating a rice bran component.
Furthermore, the present invention not only provides a novel cycloartane-type triterpene compound having an excellent carcinogenesis-preventing effect, but also provides a new method for producing a cycloartane-type triterpene compound useful as a therapeutic agent for autonomic dysfunction. It has an important meaning in that it does. That is, according to claim 3 of the present invention, a cycloartane-type triterpene compound is obtained by performing microbial conversion with filamentous fungi using a compound obtained by treating rice bran ingredients as a substrate.

Cycloartane triterpene compounds can be obtained by microbial conversion by filamentous fungi using a compound obtained by treating rice bran ingredients as a substrate. This is because of the possibility of producing safer cycloartane-type triterpene compounds, mass culture of filamentous fungi, culture of transformants incorporating genes related to conversion reactions, and related enzymes of conversion reactions extracted from filamentous fungi. This suggests the possibility of mass production by use.
Here, examples of the filamentous fungi that can be used include the genus Glomerella, the genus Mortierella, the genus Chaetomium, and the genus Aspergillus. Among the genus Glomerella, Glomerella fusarioides are more preferable.

  According to the present invention, a carcinogenic preventive agent that is safe and excellent in carcinogenic preventing effect is provided.

Next, the present invention will be described specifically by way of examples. That is, the production, isolation, identification and structural analysis of a compound having a carcinogenic effect by the microbial conversion reaction according to the present invention, and the effect confirmation test of the carcinogenic preventive according to the present invention will be described.
[Preparation of substrate]
Cycloartenol (hereinafter also referred to as “Compound 1”), 24-methylenecycloartanol (hereinafter referred to as “Compound 2” in the present specification), which is a substrate for the microbial conversion reaction. And the preparation of cycloartenone (hereinafter sometimes referred to as “compound 3” in this specification).

First, from γ-oryzanol, based on the method described in the literature (L. J. Good, T. Akihisa, “Analysis of Stereos”, Blackie Academic & Professional, London, 292, 1997), a fractional recrystallization method, Cycloartenol ferrate and 24-methylenecycloartanol ferrate were prepared. Thereafter, cycloartenol ferrate and 24-methylenecycloartanol ferrate are subjected to alkaline hydrolysis (5% potassium hydroxide / methanol solution; heating under reflux; 3 hours), respectively, so that cycloartenol (compound 1) and 24- Methylenecycloartanol (Compound 2) was prepared. Cycloartenone (Compound 3) was prepared by chemically oxidizing the hydroxyl group at the C ₃ position of Compound 1 with CrO ₃ / C ₅ H ₅ N.

[Preparation of conversion reaction cells]
The preparation of the filamentous fungus Glomerella fusarioides used in the microbial conversion reaction will be described. First, the filamentous fungus Glomerella fusarioides, more specifically in this example, Glomerella fusarioides IFO8831 (Fermentation Laboratories Foundation) in 400 mL of potato dextrose liquid medium (PDB, manufactured by Nissui Pharmaceutical Co., Ltd.) at room temperature for 5 days. Culture was performed. Furthermore, the newly prepared PDB3L was inoculated and mass-cultured at room temperature for 3 days. After completion of the culture, the cells were washed with water to prepare a conversion reaction cell used for the microorganism conversion reaction.

  In the present invention, the culture method in the step of preparing the conversion reaction cells is not particularly limited as long as it is a culture method usually used for mass culture of microorganisms. For example, the above-mentioned stirring culture method and shaking culture method are used. And aeration culture method. Any of the culture methods mentioned here are preferably carried out under aerobic conditions. For industrial culture, the aeration stirring culture method is suitable.

[Microbial conversion reaction]
The substrate was dissolved in dimethyl sulfoxide and added to pure water to prepare a reaction solution. Conversion reaction cells were added to this reaction solution, and aeration culture was performed at room temperature for 10 days.
[Isolation of compounds]
After completion of the reaction, the reaction solution was extracted with ethyl acetate. This extract was fractionated by silica gel column chromatography and octadecyl silica (ODS) column preparative high performance liquid chromatography (HPLC). The preparative HPLC column used in this example is Pegasil ODS (manufactured by Senshu Kagaku Co., Ltd., packing: particle size 5 μm, column: length 25 cm × inner diameter 10 mm).

[Microbial conversion reaction of cycloartenol]
Microbial conversion reaction was performed by the above method using cycloartenol (507 mg) as a substrate, and an ethyl acetate extract (536 mg) was obtained from the reaction solution. The following details the fractionation of this ethyl acetate extract by silica gel column chromatography and the isolation of the conversion product by preparative HPLC.
By silica gel column chromatography, Fr. 1 (eluent, n-hexane: ethyl acetate = 95: 5; 11 mg), Fr. 2 (n-hexane: ethyl acetate = 95: 5; 42 mg), Fr. 3 (n-hexane: ethyl acetate = 95: 5; 304 mg), Fr. 4 (n-hexane: ethyl acetate = 95: 5; 19 mg), Fr. 5 (n-hexane: ethyl acetate = 9: 1; 40 mg), Fr. 6 (n-hexane: ethyl acetate 4 = 1: 1; 21 mg), Fr. 7 (ethyl acetate; 9 mg), Fr. 8 (methanol; 55 mg) was obtained.

  Preparative HPLC [developing solution: methanol-water-acetic acid (95: 5: 1), 3.0 mL / min] was performed, and Fr. 2 to cycloartenone (compound 3; 24.7 mg; retention time 28.8 minutes), Fr. 4 to cycloartane-3β, 24,25-triol (compound 4; 4.8 mg; retention time 22.4 minutes), Fr. Cycloalto-25-ene-3β, 24-diol (compound 5; 4.1 mg; retention time 23.2 minutes) was isolated from 5 (see FIG. 1). In addition, cycloartenol (compound 1) which is an unreacted substrate is Fr. It was recovered as 3 components.

[Microbial conversion reaction of 24-methylenecycloartanol]
Using 24-methylenecycloartanol (500 mg) as a substrate, a microbial conversion reaction was carried out by the above method, and an ethyl acetate extract (442 mg) was obtained from the reaction solution. The following details the fractionation of this ethyl acetate extract by silica gel column chromatography and the isolation of the conversion product by preparative HPLC.

  By silica gel column chromatography, Fr. 1 (eluent, n-hexane: ethyl acetate = 95: 5; 25 mg), Fr. 2 (n-hexane: ethyl acetate = 95: 5; 355 mg), Fr. 3 (n-hexane: ethyl acetate = 95: 5; 23 mg), Fr. 4 (n-hexane: ethyl acetate = 95: 5; 5 mg), Fr. 5 (n-hexane: ethyl acetate = 9: 1; 12 mg), Fr. 6 (n-hexane: ethyl acetate = 9: 1; 19 mg), Fr. 7 (n-hexane: ethyl acetate = 8: 2; 9 mg), Fr. 8 (n-hexane: ethyl acetate = 1: 1; 6 mg), Fr. 9 (ethyl acetate; 8 mg), Fr. 10 (methanol; 31 mg) was obtained.

Of these, preparative HPLC [developing solution: methanol-acetic acid (100: 1), 3.0 mL / min] was performed, and Fr. 2 to cycloeucarenol (compound 6; 4.8 mg; retention time 27.2 minutes), 24-methylcycloartane-3β, 24,24 ¹ -triol (compound 7; 3.7 mg; retention time 8.4 minutes) , 24 ¹ -methoxy-24-methylcycloartane-3β, 24-diol (compound 8; 6.5 mg; retention time 18.4 min) was isolated. Fr. 3 to cycloeucarenol (compound 6; 4.8 mg; retention time 27.2 minutes), 24 ¹ -methoxy-24-methylcycloartane-3β, 24-diol (compound 8; 6.5 mg; retention time 18.4) Min) was isolated. In addition, preparative HPLC [developing solution: methanol-water-acetic acid (95: 5: 1), 3.0 mL / preparative] Cycloalto-25-ene-3β, 24-diol (compound 5; 3.6 mg; retention time 26.8 minutes) was isolated from 5 (see FIG. 2). Note that Fr. In Fig. 2, 24-methylenecycloartanol (compound 2) which is an unreacted substrate is Fr. It was recovered as two components.

[Microbial conversion of cycloartenone]
Microbial conversion reaction was performed by the above method using cycloartenone (568 mg) as a substrate, and an ethyl acetate extract (546 mg) was obtained from the reaction solution. The following details the fractionation of this ethyl acetate extract by silica gel column chromatography and the isolation of the conversion product by preparative HPLC. By silica gel column chromatography, Fr. 1 [eluent, n-hexane: ethyl acetate = 95: 5; 309 mg], Fr. 2 [n-hexane: ethyl acetate = 8: 2; 32 mg], Fr. 3 (n-hexane: ethyl acetate = 1: 1; 130 mg), Fr. 4 (ethyl acetate; 30 mg), Fr. 5 (methanol; 107 mg) was obtained.

  Preparative HPLC [developing solution: methanol-water-acetic acid (95: 5: 1), 3.0 mL / min] was performed, and Fr. 1 to cycloartane-3,24-dione (compound 9; 3.1 mg; retention time 16.2 minutes), 24-hydroxycycloalto-25-en-3-one (compound 11; 5.2 mg; retention time) 13.8 minutes), 24,25-dihydroxycycloartan-3-one (compound 13; 5.3 mg; retention time 8.7 minutes), 25-hydroxy-24-methoxycycloartan-3-one (compound 15; 42 mg; retention time 12.9 minutes) was isolated. Fr. 2 to 4,4,14-trimethyl-9,19-cyclopregnan-3,20-dione (compound 10; 3.5 mg; retention time 6.9 minutes), compound 11 (5.2 mg; retention time 13 8 minutes), cycloalto-25-ene-3,24-dione (compound 12; 3.0 mg; retention time 19.5 minutes) was isolated. Fr. 3 isolated compound 13 (5.3 mg; retention time 8.7 minutes), 25-hydroxycycloalto-23-en-3-one (compound 14; 3.9 mg; retention time 16.5 minutes) (See FIG. 3). The reaction substrate cycloartenone is Fr. It was recovered as one component.

[Structural analysis and identification of compounds 4 to 15]
The structural analysis and identification of 12 kinds of compounds 4 to 15 isolated as described above will be described. Structural analysis of compounds 4 to 15 is MS, IR, ¹ H-NMR, ¹³ C-NMR, and correlation spectroscopy ( ¹ H- ¹ HCOSY), which is a two-dimensional NMR method, heteronuclear multiquantum coherence method (HMQC), Heterogeneous nuclear remote multiquantum correlation (HMBC) and nuclear overhauser exchange spectroscopy (NOESY) were used. Chemical structural formulas of 12 kinds of compounds 4 to 15 subjected to structural analysis are shown in FIGS.

Of these, four types of compounds 7, 8, 10, and 15 are novel compounds that have not been described so far. ¹ H-NMR and ¹³ C-NMR of novel compounds 7, 8, ¹⁰ , and 15 are shown in Tables 1 to 4 together with HMBC data. FIG. 4 shows the structural formulas showing the carbon numbers of the novel compounds 7, 8, 10, and 15. FIG. 5 shows the nuclear overhauser effect (NOE) correlation obtained by the NOESY spectral method of these novel compounds. Is indicated by a double-headed arrow together with the three-dimensional structural formula. These NOE correlations are consistent with the structures assigned for compounds 7, 8, 10, and 15.

The structures of known compounds 4 to 6, 9, and 11 to 14 were confirmed by comparing various spectral values of these compounds with literature values (shown below).
Compound 4: Inada et al. Phytochemistry 46, 2, 379-381, 1997 Compound 5, 11: Cabrera et al. Journal of Natural Products 59, 343-347, 1996 Compound 6: Akihisa et al. Physochemistry 47, 1107-1110, 1998 Compound 9, 13: Inada et al. Journal of Natural Products 58, 1143-1146, 1995 Compound 12, 14: DePascal et al. Phytochemistry 26, 1767-1776, 1987. It is not known about the carcinogenic preventive effect of these compounds.

Properties and spectral data of the new compounds 7, 8, 10, and 15 are shown below.
(1) 24-Methylcycloartane-3β, 24,24 ¹ -triol (Compound 7)
Colorless crystals. Melting point: 175-177 ° C. Specific rotation [α] ²⁵ _D : +25.50 (concentration c = 0.10 (g / 100 mL), solvent chloroform). IR v _max : 3407, 2928 cm ^-1
EI-MS (m / z value, (relative ionic strength)): 474 (8) [M] ⁺ , 456 (30) [M−H ₂ O] ⁺ , 441 (23) [m / z 456-CH ₃ ] 423 (30) [m / z 441-H ₂ O] ⁺ , 334 (23), 203 (32), 175 (57), 95 (100). High resolution EI-MS (m / z value): 474.644 (theoretical value C ₃₁ H ₅₄ O ₃ [M−H ₂ O] ⁺ 474.4073).
¹ H-NMR and ¹³ C-NMR data are shown in Table 1 together with HMBC data.
¹³ C-NMR (150 MHz), ¹ H-NMR (600 MHz) and HMBC spectrum data (solvent: CDCl ₃ ) of Compound 7

(2) 24 ¹ -methoxy-24-methylcycloartane-3β, 24-diol (Compound 8)
Colorless crystals. Melting point: 168-171 ° C. Specific rotation [α] ²⁵ _D : + 4.3 ° (concentration c = 0.10 (g / 100 mL), solvent chloroform). IR v _max : 3409, 2938 cm ⁻¹ .
EI-MS (m / z value, (relative ionic strength)): 488 (13) [M] ⁺ , 470 (45) [M−H ₂ O] ⁺ , 457 (100), 438 (37) [m / _{z470-HOCH 3], 423 (} 52) [m / z438-CH 3], 317 (83), 175 (44), 95 (68).
High resolution EI-MS (m / z value): 488.4228 (theoretical value C ₃₁ H ₅₆ O ₃ [M] ⁺ 488.4229).
¹ H-NMR and ¹³ C-NMR data are shown in Table 2 together with HMBC data.
¹³ C-NMR (150 MHz), ¹ H-NMR (600 MHz) and HMBC spectrum data (solvent: CDCl ₃ ) of Compound 8

(3) 4,4,14-trimethyl-9,19-cyclopregnan-3,20-dione (compound 10)
Colorless crystals. Melting point: 165-168 ° C. Specific rotation [α] ²⁵ _D : +8.30 (concentration c = 0.16 (g / 100 mL), solvent chloroform). IR v _max : 2931, 1710 cm ⁻¹ .
EI-MS (m / z value, (relative ionic strength)): 356 (100) [M] ⁺ , 341 (52) [M-CH ₃ ] ⁺ , 313 (52) [m / z 341 -CO], 271 (26) [m / z 313-C ₃ H ₆ ], 218 (100) [m / z 271-C ₄ H ₆ ], 175 (70), 137 (50), 121 (47), 107 (60), 95 (68).
High resolution EI-MS (m / z value): 356.2714 (theoretical C ₂₄ H ₃₆ O ₂ [M] ⁺ 356.2715).
¹ H-NMR and ¹³ C-NMR data are shown in Table 3 together with HMBC data.
¹³ C-NMR (150 MHz), ¹ H-NMR (600 MHz) and HMBC spectrum data (solvent: CDCl ₃ ) of Compound 10

(4) 25-hydroxy-24-methoxycycloartan-3-one (compound 15)
Colorless crystals. Melting point: 198-199 ° C. Specific rotation [α] ²⁵ _D : +16.40 (concentration c = 0.18 (g / 100 mL), solvent chloroform). IR v _max : 3445, 2931, 1710 cm ⁻¹ .
EI-MS (m / z value, (relative ionic strength)): 472 (15) [M] ⁺ , 440 (45) [M-HOCH ₃ ] ⁺ , 313 (50) [M-side chain (C ₉ H ₁₉ O ₂ )] ⁺ 271
(5), 73 (100).
High resolution EI-MS (m / z value): 472.3924 (theoretical value C ₃₁ H ₅₂ O ₃ [M] ⁺ 472.3916)
¹ H-NMR and ¹³ C-NMR data are shown in Table 4 together with HMBC data. ¹³ C-NMR (150 MHz), ¹ H-NMR (600 MHz) and HMBC spectrum data of the compound 15 (solvent: CDCl ₃ )

The structure determination of novel compounds 7, 8, 10, and 15 will be described below (see FIG. 4).
Compound 7 was shown to have the molecular formula C ₃₁ H ₅₄ O ₃ from high resolution EI-MS ([M] ⁺ m / z 474.4064) and ¹³ C-NMR. In addition, this compound has a methylene group [δ _H 0.33 (d, J = 4.4 Hz), 0.55 (d, J = 4.2 Hz)] from IR spectrum, ¹ H-NMR, ¹³ C-NMR. Was indicated, suggesting the presence of a cyclopropyl group. In addition, four tertiary methyl groups [δ _H 0.81, 0.89, 0.97, 0.97 (each s)] and one secondary methyl group [δ _H 0.90 (d, J = 7.3 Hz)], inpropyl group [δ _H 0.93 (d, J = 6.3 Hz), 0.94 (d, J = 6.8 Hz)], and one hydroxymethine group [3407 cm ⁻¹ ; δ _H 3.28 (m); δ _C 78.8], one hydroxymethylene group [δ _H 3.47 (d, J = 11.3 Hz), 3.62 (d, J = 10 .7 Hz); δ _C 65.9]. In EI-MS, m / z 456 corresponds to an ion due to desorption of H ₂ O, m / z 441 corresponds to (m / z 456-CH ₃ ), and m / z 423 corresponds to (m / z 441-H ₂ O). Ions were observed. Compared with these values and literature values of cycloartane compounds (De Pascual et al., Phytocchemistry 26, 1767-1776, 1987), compound 7 has a 24-methylcycloartane-3β, 24,24 ¹ -triol structure. It became clear to have. The correctness of this structure was confirmed by analysis of ¹ H- ¹ HCOSY, HMQC, HMBC, and NOESY spectra.

Compound 8 was shown to have the molecular formula C ₃₂ H ₅₆ O ₃ from high resolution EI-MS ([M] ⁺ m / z 488.4229) and ¹³ C-NMR. In addition, this compound has a methylene group [δ _H 0.33 (d, J = 4.4 Hz), 0.55 (d, J = 4.2 Hz)] from IR spectrum, ¹ H-NMR, ¹³ C-NMR. Was indicated, suggesting the presence of a cyclopropyl group. In addition, four tertiary methyl groups [δ _H 0.81, 0.90, 0.97, 0.97 (each s)] and one secondary methyl group [δ _H 0.90 (d, J = 6.5 Hz)], inpropyl group [δ _H 0.92 (d, J = 6.3 Hz), 0.94 (d, J = 6.8 Hz)], and one hydroxymethine group [3409 cm ⁻¹ ; δ _H 3.29 (m); δ _C 78.9], suggesting the presence of one methoxyl group [δ _H 3.23 (m); δ _C 49.4]. In EI-MS, ions due to the elimination of H ₂ O were observed at m / z 470, and ions from which HOCH ₃ was eliminated due to cleavage of C ₂₄ -C ₂₄ ¹ were observed at m / z 438. These were compared with the spectrum value of Compound 7, and it was found that Compound 8 has a 24 ¹ -methoxy-24-methylcycloartane-3β, 24-diol structure. The correctness of this structure was confirmed by analysis of ¹ H- ¹ HCOSY, HMQC, HMBC, and NOESY spectra.

Compound 10 was shown to have the molecular formula C ₂₄ H ₃₆ O ₂ by high resolution EI-MS ([M] ⁺ m / z 356.2714) and ¹³ C-NMR. In addition, this compound has a methylene group [δ _H 0.57 (d, J = 4.6 Hz), 0.82 (d, J = 4.3 Hz)] from IR spectrum, ¹ H-NMR, ¹³ C-NMR. Was indicated, suggesting the presence of a cyclopropyl group. Four more tertiary methyl groups [δ _H 0.93, 0.98, 1.05, 1.10, 2.12 (each s)], and two carbonyl groups [1710 cm ⁻¹ ; δ _C 210.3, 216.4] was suggested. In EI-MS, a cleaved ion in which the C ₂₁ methyl group was eliminated by cleavage of C ₂₀ -C ₂₁ at m / z 341 and an ion in which CH ₃ CO was eliminated by cleavage of C ₁₇ -C ₂₀ at m / z 313 were observed. It was. Compared to these spectral values and the literature values of cycloartane compounds (Barik et al., Physochemistry 35, 1001-1004, 1994), compound 10 is 4,4,14-trimethyl-9,19-cyclopregnane. It became clear that it had a -3,20-dione structure. The correctness of this structure was confirmed by analysis of ¹ H- ¹ HCOSY, HMQC, HMBC, and NOESY spectra.

Compound 15 was shown to have the molecular formula C ₃₁ H ₅₂ O ₃ from high resolution EI-MS ([M] ⁺ m / z 472.3924) and ¹³ C-NMR. In addition, this compound has a methylene group [δ _H 0.57 (d, J = 4.2 Hz), 0.79 (d, J = 3.7 Hz)] from IR spectrum, ¹ H-NMR, ¹³ C-NMR. Was indicated, suggesting the presence of a cyclopropyl group. In addition, four tertiary methyl groups [δ _H 0.91, 1.00, 1.05, 1.10, (each s)] and one secondary methyl group [δ _H 0.89 (d , J = 6.4 Hz)], inpropyl group [δ _H 1.10, 1.13 (each s), and hydroxyl group [3445 cm ⁻¹ ], carbonyl group [1710 cm ⁻¹ ; δ _C 213.0], The presence of a methoxyl group (δ _H 3.23 (s); δ _C 49.1) was suggested. In EI-MS, an ion due to CH ₃ OH elimination at C ₂₄ position at m / z 440, a cleavage ion from which side chain (C ₉ H ₁₉ O ₂ ) is eliminated due to cleavage of C ₁₇ -C ₂₀ at m / z 313, A cleaved ion of D ring with 1H due to bond cleavage of C ₁₃ -C ₁₇ and C ₁₄ -C ₁₅ was observed at m / z 271. By comparing these values with the literature values of cycloartane compounds (Inada et al., Physochemistry 46, 379-381, 1997), the spectral value of compound 13 indicates that compound 15 is 25-hydroxy-24-methoxycycloartane- It became clear that it had a 3-one structure. The correctness of this structure was confirmed by analysis of ¹ H- ¹ HCOSY, HMQC, HMBC, and NOESY spectra.

Compounds 7 and 8 may be those obtained by chemical conversion reaction of 24-methylenecycloartanol (Compound 2). A method for synthesizing compounds 7 and 8 using a chemical conversion reaction is shown below (see FIG. 5).
[Preparation of Compound 7 by Chemical Method]
(1) Compound 2 (100 mg) was dissolved in methylene chloride, sodium bicarbonate (250 mg) and m-chloroperbenzoic acid (500 mg) were added, and the mixture was stirred at room temperature for 48 hours.
(2) After completion of the reaction, the reaction solution was washed with a 1N aqueous sodium hydroxide solution, and then the methylene chloride layer was washed with water.

(3) Preparative HPLC:; do [eluent methanol flow rate 3.0 mL / min], 24, 24 ¹ - epoxycycloalkyl Altan -3β- ol (Compound 16; retention time 16.8 min; 31.6 mg) Obtained.
(4) Compound 16 (8.7 mg) was dissolved in 1,2-dimethoxyethane, an aqueous perchloric acid solution (2 mL) was added, and the mixture was stirred at room temperature for 16 hours.
(5) After completion of the reaction, extraction was performed with diethyl ether, and the ether layer was washed with 1M sodium carbonate and subsequently with water. The ether extraction section was subjected to preparative HPLC [eluent: methanol; flow rate: 3.0 mL / min] to obtain Compound 7 (2.6 m; retention time 12.9 minutes).

The above-described cycloartane triterpene compounds of the present invention can be used alone or in combination.
Further, the cycloartane type triterpene compound of the present invention can be made into a pharmaceutical in combination with an appropriate pharmaceutical carrier or diluent, and can be formulated by any usual method for oral or parenteral administration. It can be formulated into a solid, semi-solid or liquid dosage form. In prescribing, it is good also as a compounding agent with another pharmaceutical active ingredient.

  For example, various preparations described in the Japanese Pharmacopoeia, that is, solid preparations for internal use such as tablets, pills, capsules, granules, powders, dry extracts, troches, flow extracts, elixirs, spirits It can be formulated into liquid preparations such as syrups and limonades, external liquids such as tinctures, liniments and lotions, and external preparations such as plasters, ointments and poultices. If administration is possible, inhalants, aerosols, injections, eye drops, suppositories and the like may be formulated according to the intended use.

In oral administration, it is preferable to administer to adults in the range of 0.5 to 500 mg / kg body weight.
In addition, the cycloartane triterpene compound of the present invention is not only used as a carcinogenic agent but also useful for producing functional foods and drinks that are effective in preventing carcinogenesis when added to various foods and drinks. is there. Examples of the form of such functional foods and drinks include granules, tablet confectionery, jelly, strawberries, beverages and the like. Furthermore, the cycloartane-type triterpene compound of the present invention can be used not only as a food eaten by humans but also added to feeds of animals other than humans such as pets, livestock, and sport animals.

[Preparation of Compound 8 by Chemical Method]
(1) to (3) Compound 16 was obtained in the same manner as (1) to (3) above.
(4) Compound 16 (8.7 mg) was dissolved in 1,2-dimethoxyethane, perchloric acid / methanol (2 mL) was added, and the mixture was stirred at room temperature for 16 hours.
(5) After completion of the reaction, extraction was performed with diethyl ether, and the ether layer was washed with 1M sodium carbonate and subsequently with water. The ether extract was subjected to preparative HPLC [eluent: methanol; flow rate: 3.0 mL / min] to obtain Compound 8 (1.7 mg; retention time 18.3 minutes).

[Confirmation test for carcinogenesis prevention effect]
Next, tests conducted to confirm the effects of the present invention will be described.
(Epstein-Barr virus (EBV) activation inhibition test)
In the Raji strain, a cultured cell derived from Burkitt lymphoma that contains the EBV genome, many of the compounds that inhibit the expression of the EBV genome have been tested in a two-stage mouse skin carcinogenesis experiment. We focused on the fact that it acts as a tumor promoter. Then, a virus / genome inactivating substance that inhibits the expression of EBV / genome was searched for from the conversion product obtained from the microbial conversion reaction of cycloartane triterpenoids obtained from rice bran extract by filamentous fungi. This method, which focuses on EBV genome expression inhibitory action, is based on Raji strain culture system, TPA (tetradecanoyl phorbol acetate), which is an oncogenic promoter, and n-butyric acid that works synergistically for activity expression. In this method, a test substance is added and cultured, and the detection is performed by the indirect fluorescent antibody method using an antibody derived from a cell activated by TPA. This method is excellent in that it is rapid and excellent in quantitativeness, and in addition, it can detect a trace amount of active ingredient.

First, the test procedure will be described with reference to the scheme of FIG. This procedure is based on the method of Tokuda et al. (Cancer Letters, 40, 309, 1998).
(1) To a 1 × 10 ⁶ / mL radio cell, 32 pmo1 of 12-O-tetradecanoylphorbol-13-acetate (TPA) at a concentration of 20 ng / mL was added as a tumor promoter, and TPA activity expression was further increased. Therefore, n-butyric acid which acts as a synergistic effect was added.
(2) A predetermined amount of a test substance dissolved in water, ethanol, or dimethyl sulfoxide was added thereto, and cultured at 37 ° C. for 48 hours.
(3) After completion of the culture, expression of EBV early antigen was detected by an indirect fluorescent antibody method using serum from patients with nasopharyngeal cancer (including antibodies derived from cells activated by TPA).

After performing the test according to the above procedure, the expression rate of the EBV early antigen in the test substance addition group was determined with the expression rate of the EBV early antigen in the group to which only TPA was added (control) as 100%. Was used to calculate the EBV early antigen expression inhibition rate (%) of the test substance.
EBV early antigen expression inhibition rate (%) =
EBV early antigen expression rate (%) in 100-test substance addition group: Formula (1)
In the assay, as described above, compounds 1 to 15 obtained from the conversion reaction of cycloartane triterpenoids by the filamentous fungus Glomerella fusarioides were used as test substances, and each test substance was prepared at various concentrations (test substance / TPA molar ratio). I went. The results of this test are shown in Table 5.
EBV early antigen expression inhibition rate of cycloartane type triterpene (compounds 1 to 15) (%)

The numerical value in parentheses in the column where the concentration of the test substance is 1000-fold molar concentration indicates the survival rate (%) of the raji cells. It can be said that the higher the value, the smaller the adverse effect on normal cells, that is, the higher the safety. As shown in Table 5, the inhibition rate of these compounds is 28 to 37% at a 100-fold molar concentration with respect to TPA, and has high activity. In particular, 24-methylcycloanolane-3β, 24,24 ¹ -triol (compound 7) and 24 ¹ -methoxy-24-methylcycloartane-3β, 24-diol (compound 8) are 15% at a 10-fold molar concentration. It has been confirmed that it has very high activity as described above and is excellent in anti-tumor promoter activity. In addition, this inhibition rate is a vitamin A derivative, and β-carotene (Murakami et al., Biosci. Biotech. Biochem., Vol. 60, page 1, 1996) whose carcinogenesis-preventing effect has been confirmed in various animal experiments. Also showed a high value. Furthermore, since these showed a high survival rate in the present assay, they can be expected as carcinogenic preventive agents with high safety.

Furthermore, in order to test the effect of the present invention, compounds 1 to 15 were tested for inhibition against NO (nitrogen monoxide) damage and showed activity, which will be described.
(Cell shape change suppression test by NOR1 treatment)
Cell shape change suppression test by (±)-(E) -4-methyl-2-[(E) -hydroxyimino] -5-nitro-6mumethoxy-3-hexenamide (NOR1) treatment is a primary oncogenic initiator. This is a test known as a screening method. NO is a gaseous free radical, and it is said that the risk of gastric cancer and colon cancer increases when its overproduction becomes chronic. From this, it can be said that suppressing excessive NO production is effective in preventing carcinogenesis. This test is excellent in that it allows rapid detection of trace active ingredients.

The test method will be described. This procedure is based on the method of Akiku et al. (Cancer Letters 205, 9-13, 2004).
(1) 5 × 10 ⁵ / mL human normal liver-derived cells obtained from Eagle's minimum required medium are cultured for 3 days.
(2) A test substance is added thereto, and after 1 minute, NOR as a NO donor is added and cultured in a CO ₂ incubator.
(3) The change in cell morphology is measured with an optical microscope. (Measure the number of cells that showed more than 250 morphological changes.)

After conducting the test according to the above procedure, the group to which the test substance was not added was set as a control, and the NO productivity suppression rate by the NOR1 treatment was calculated by the following formula.
Inhibition rate of cell shape change by NOR1 treatment = cells whose shape changed only by NOR1 (%) / cells whose shape changed when NOR1 and a test substance were added (%) (2)

Table 6 shows the results of the test conducted using the compounds (1 to 15) obtained from the conversion reaction of cycloartane triterpenoids by the filamentous fungus Glomerella fusarioides as described above as test substances. As a reference compound, curcumin (Cancer Letters 159, 135, 2000), which is known to have a carcinogenic effect, and NO scavenger 2- (4-carboxyphenyl) -4,4,5,5-tetra The value of methylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) is also shown.
Inhibition rate of cycloartane-type triterpenes (compounds 1 to 15) against NO damage

  As shown in Table 6, Compounds 7 and 8 showed the same activity as the reference compound curcumin. From these results, it was confirmed that these compounds are excellent in anti-carcinogenic initiator activity.

It is a figure which shows the chemical structural formula of the microbial conversion reaction product (compounds 3-5) of cycloartenol (compound 1) by a filamentous fungus. It is a figure which shows the chemical structural formula of the microorganisms conversion reaction product (compounds 5-8) of 24-methylenecycloartanol (compound 2) by a filamentous fungus. It is a figure which shows the chemical structural formula of the microbial conversion reaction product (compounds 9-15) of cycloartenone (compound 3) by a filamentous fungus. It is a figure which shows the chemical structural formula of the novel compounds 7, 8, 10, and 15. It is a figure explaining the nuclear overhauser effect (NOE) correlation obtained by the NOESY spectrum method of the novel compounds 7, 8, 10, and 15 concerning this invention. It is a figure which shows the method of synthesize | combining the compounds 7 and 8 chemically. It is a figure explaining the procedure of an EBV activation suppression test.

Claims (3)

Cycloalkyl Yu Carre Nord, 24 methylcyclopentadienyl Altan-3.beta, 24, 24 ¹ - triol, 24 ¹ - methoxy-24-methyl-cyclopropyl Altan-3.beta, 24-diol, cycloalkyl Altan -3,24- dione, 4,4,14 -Trimethyl-9,19-cyclopregnan-3,20-dione, 24,25-dihydroxycycloartan-3-one, 25-hydroxycycloalto-23-en-3-one, 25-hydroxy-24-methoxy A cancer preventive agent comprising at least one cycloartane-type triterpene compound selected from the group consisting of cycloartan-3-one.   The cycloartane-type triterpene compound is a reaction product obtained from a microbial conversion reaction by a filamentous fungus using a compound obtained by treating a rice bran component as a substrate, or a chemical conversion reaction of a compound obtained by treating a rice bran component The carcinogenesis-preventing agent according to claim 1, which is a reaction product obtained by   A method for producing a cycloartane-type triterpene compound, comprising obtaining a cycloartane-type triterpene-type compound by performing microbial conversion with a filamentous fungus using a compound obtained by treating rice bran ingredients as a substrate.

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